Process of microbic biotechnology for completely degrading gluten in flours

ABSTRACT

The present invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation from both bread and durum wheat, barley, rye and oat flour. In particular, the invention concerns the use of lactic acid bacteria selected and fungal enzymes for the gluten complete degradation (residual gluten concentration lower than 20 ppm) of cereal flours, which after detoxification can be used according to a standardized biotechnological protocol for the production of various gluten-free foods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.13/131,458, filed Oct. 13, 2011, which is a National Stage entry ofInternational Application No. PCT/IT2009/000569, filed Dec. 17, 2009,which claims priority to Italian Patent Application No. RM2008A000690,filed Dec. 23, 2008. The disclosures of the prior applications arehereby incorporated in their entirety by reference.

The present invention concerns a microbic biotechnology for completelydegrading gluten in flours. Particularly, the process according to theinvention involves the use of selected lactic bacteria and fungalproteases routinely used for the manufacture of leavened baked goods,under liquid fermentation conditions for the complete gluten degradation(residual gluten concentration lower than 20 ppm). Cereal floursresulting from fermentation process can be used as raw material for theproduction of gluten-free foods designed to be eaten by celiac patients.Proposed biotechnological process results in various economic, social,nutritional and sensory advantages compared to current productionprocess of gluten-free foods made by ingredients which are naturallygluten-free or as result of extraction processes.

Gluten intolerance epidemiology or celiac disease is continuouslygrowing. Last surveys about European and the United States populationreport an incidence of 1/100 affected individuals (Rewers, 2005.Epidemiology of celiac disease; what are the prevalence, incidence, andprogression of celiac disease. Gastroenterology 128:47-51). According tocurrent knowledge, unique effective therapeutic remedy against thisalimentary intolerance is a completely gluten-free diet to be observedrigorously for all the life (Hamer, 2005. Celiac Disease: Background andbiochemical aspects. Biotechnol Advanc 23:401-408). It is known, forexample, the use of lactic bacteria for the preparation of baked goodsfrom gluten-free flours (More et al. Cereal Chemistry, AmericanAssociation of Cereal Chemists. Minneapolis, US, vol. 84, no. 4, 1 Jan.2007, pp. 357-364 and Moore et al., European Food Research andTechnology, vol. 226, 6 Jun. 2007, pp. 1309-1316). However, thegluten-free diet results also in apparent disadvantages. It is veryexpensive, gluten-free products, when compared to cereal based products,display lower sensory quality and storage, the diet is difficult to bestrictly observed and it needs to be continuously monitored bydieticians, also taking in consideration nutritional imbalances (forexample fibres, minerals and vitamins) resulting from complete absenceof cereals in the feeding (Grehn et al., 2001. Dietary habits of Swedishadult coeliac patients treated by a gluten-free diet for 10 years. ScandJ Nutr 45: 178-182; Mariani et al., 1998. The gluten-free diet: anutritional risk factor for adolescents with celiac disease. J PediartGastroenterol Nut 27: 519-523; Thompson et al., 2005. Gluten-free dietsurvey: are Americans with celiac disease consuming recommended amountsof fibre, iron, calcium and grain foods? J. Human. Nutr. Diet.18:163-169). Moreover, in some cases (for example “refractory sprue”)also the complete observance of gluten-free diet does not allow acomplete recovery of the intestinal functionality (Sollid and Khosla,2004. Future therapeutic options for celiac disease. Gastroenterol.Hepatol. 2:140-147). Within gluten-free diet alternative therapeutictreatments, various studies have taken advantage of current knowledge onthe sequences of toxic epitopes and have considered the use of microbialenzyme, particularly prolyl-endopeptidase (PEPs), for the hydrolysis ofthese polypeptides. Microbial enzymes have been suggested as dietsupplements (Shan et al., 2004. Comparative biochemical analysis ofthree bacterial prolyl-endopeptidases: implications for celiac sprue.Biochem. J. 383:311-318) and/or for gluten in vitro detoxification (Chenet al., 2003. Identification and characterization of Lactobacillushelveticus PePO2, an endopeptidase with post-proline specificity. Appl.Environ. Microbiol. 69:1276-1282; Stepniak et al., 2005. Highlyefficient gluten degradation with a newly identifiedprolyl-endoprotease: implications for celiac disease. Am. J. Physiol.Gastrointest. Liver Physiol. 291:G621-G629).

Patent application WO2008/010252 and Cagno et al. (Journal of FoodProtection, vol. 71, no. 7, 2008, pp. 1491-1495) disclose a process forthe preparation of baked goods from gluten-free flours and aiming toimprove the nutritional, sensory and storage characteristics of theseproducts prepared from gluten-free ingredients.

During the last few decades also the biotechnology of leavened bakedgoods is considerably changed, influencing the nutritional habits ofentire populations previously subjected to a gluten based diet.Currently, leavened baked goods are manufactured by means of extremelyrapid technological processes (for example, chemical leavening agents orusing baker's yeast), completely replacing long fermentation processesusing wild lactic acid bacteria and yeasts originated from rawmaterials, and used as “sourdoughs”. By means of current processes thecereal components (for example proteins) are not subjected to anyhydrolytic activity during food processing, maintaining the raw materialcharacteristics (Gobbetti, 1998. The sourdough microflora: interactionsbetween lactic bacteria and yeasts. Trends Food Sci. Technol.9:267-274). Based on these considerations and taking advantage of enzymepotentialities of a mixture of selected lactic bacteria, various studies(Di Cagno et al., 2002. proteolysis by sourdough lactic bacteria:effects on wheat flour protein fractions and gliadin peptides involvedin human cereal intolerance. Appl. Environ. Microbiol. 68:623-633; DiCagno et al., 2004. Sourdough bread made from wheat and non toxic floursand started with selected lactobacilli is tolerated in celiac spruepatients. Appl. Environ. Microbiol. 70:1088-1096; Di Cagno et al., 2005.Pasta made from durum wheat semolina fermented with selectedlactobacilli as a tool for a potential decrease of the glutenintolerance. J. Agr. Food Chem. 53:4393-4402; De Angelis et al., 2005.VSL#3 probiotic preparation has the capacity to hydrolyze gliadinpolypeptides responsible for celiac sprue. Biochim. Biophys. Acta.1762:80-93) demonstrated that using a traditional biotechnology, basedon the use of selected lactic acid bacteria and long fermentation times,it is possible to reduce remarkably the initial cereal glutenconcentration.

Codex Alimentarius, adopted by WHO (World Health Organization) and FAO(Food and Agricultural Organization), distinguishes “gluten-freeproducts”, containing ingredients with gluten concentration lower than20 ppm, and “gluten-free made products”, having a residual glutenconcentration lower than 200 ppm. However, various studies, culminatedwith guidelines as issued by “Prolamins Working Group”, suggest, in anycase, a gluten threshold lower than 20 ppm to be maintained (Stern etal., 2001. Analysis and clinical effects of gluten in celiac disease.Eur. J. Gastroenterol. Hepatol. 13:741-747). A recent study by Rizzelloet al. (Rizzello et al., 2007. Highly efficient gluten degradation bylactobacilli and fungal proteases during food processing: newperspectives for celiac disease. Appl. Environ. Microbiol. 73:4499-4507)considered the use of a more complex mixture consisting of 10 selectedlactic acid bacteria, fungal proteases and long fermentation times (48 hat 37° C.), under semi-liquid kneading conditions. According toelectrophoretic, chromatographic and immunological analyses, the glutencontained in the wheat flour was degraded to a concentration lower than20 ppm threshold.

It is further known Patent Application WO2006/097415 wherein,analogously to above mentioned study, a process for gluten degradationby means of the use of a complex mixture consisting of at least sixlactic acid bacteria and/or bifidobacteria and long fermentation times(24-31 hours) is described. However, the method described in thisdocument is not suitable in order the gluten to be completely degraded;therefore it results the impossibility for the administration to celiacpatients. FIG. 1B of Patent Application WO2006/097415, in fact, showsthat, after hydrolysis by means of microorganisms, there are yet clearspots for not degraded gliadins and this is confirmed in table 2, againfrom the same document, from which it is apparent that while somegliadins are partially hydrolysed others are not sensitive to hydrolysisprocess.

Based on literature and previously described data, some problems appearto be of major concern for the manufacture of gluten-free foods fromdetoxified cereal flour: (i) to simplify the composition of selectedlactic bacteria to be used for the degradation process; (ii) to reduceconsiderably fermentation times thus making suitable to be used inindustrial processes; (iii) to demonstrate the ability of lactic acidbacteria and fungal enzymes in order to act effectively on bread anddurum wheat flours belonging to different varieties, and on barley, ryeand oat; (iv) to provide a biotechnological process for glutenhydrolysis allowing detoxified cereal flours for the production ofgluten-free products to be used; and (v) to demonstrate, by means of invivo chronic medical trials, the absolute tolerance for celiac patientsafter extended administration of gluten-free products based ondetoxified wheat flour.

In the light of above, it is therefore apparent the need to providematerials and methods for the preparation of gluten-free baked goodsmade of detoxified cereal flour which do not display, on one side, thedisadvantages emerged from economic, social, nutritional and sensorysurvey of literature data and, on the other hand, the disadvantages ofthe currently commercially available gluten-free products.

The Authors of the present invention now observed that using only twoselected lactic acid bacteria, in combination with fungal proteases, thefermentation times needed for gluten degradation are remarkablydecreased. Moreover, the ability of lactic acid bacteria and fungalproteases for the complete gluten degradation from different bread anddurum wheat varieties, barley, rye and oat flours was proved; abiotechnological protocol for the production of various leavened bakedgoods from detoxified wheat flour was provided; and the absolute producttolerance for celiac patients was demonstrated, thus allowing, in acompletely innovative way, the wheat flour to be used as an ingredientfor the manufacture of gluten-free leavened baked goods.

Lactic acid bacteria according to the present invention belong to theLactobacillus genus and were isolated from “sourdoughs” used for themanufacture of Southern Italy typical breads. Lactobacillussanfranciscensis DPPMA12 (deposited as DSMZ N. DSM22063 on 28 Nov. 2008)and Lactobacillus plantarum DPPMA125 (deposited as DSMZ N. DSM22064 on28 Nov. 2008).

A biotechnological protocol involving the use of selected lactic acidbacteria and fungal proteases in an extremely rapid fermentation process(12-20 h at 30-37° C.) of cereal flours re-suspended in water at 20-50%weight and successive use thereof, at various percentages according tothe desired characteristics, as an ingredient for a short leavening(about 1-3 h) by means of baker's yeast, for the production ofgluten-free leavened baked goods (residual gluten content lower than 20ppm) was standardized and optimized.

Below an outline of the biotechnological protocol for the production ofleavened baked goods from detoxified gluten-free wheat flour isreported.

According to one of possible formulations, baked goods, containing 10 gequivalent of initial gluten, were daily administered to celiac patientsover a period of 60 days. Immunochemical and histological assays provedthe absolute tolerance of preparation from detoxified gluten-free flour.

According to complementary analyses using electrophoretic,chromatographic and immunological techniques, the fermentation processby means of selected lactic acid bacteria, not used in previous studies,and fungal proteases, according to the present invention, allows: (i)complete gluten detoxification (residual gluten content lower than 20ppm); (ii) production of hydrolyzed flour consisting of a mixture of lowmolecular weight peptides and, especially, amino acids (ca. 15.000 mg/kgwith respect to <1000 mg/kg in wheat flour) which increased thenutritional features with respect to conventional gluten-free products;(iii) remarkable reduction of process times compared to literaturereported data, thus making said process suitable to an industrialscaling up transformation; (iv) production of gluten-free baked goodswith different ingredient formulations involving the use of detoxifiedwheat flour at different concentrations (20-50%); and (v) absolutetolerance of the preparation for celiac patients after extendedadministration, according to first time reported medical data.

Products obtainable according to the process of the present inventiondisplay advantageous sensory, rheological and chemical properties notoffered by prior art products (gluten-free products obtained fromnaturally gluten-free flours). Products according to the invention, infact, maintain the nutritional properties of gluten containing floursthus offering better nutritional characteristics compared to productsobtained from gluten-free flours.

Moreover, the products according to the invention are completelygluten-free as result of gluten degradation process carried out byinventive lactic acid bacteria, otherwise than products obtained usingknown lactic acid bacteria (WO2006/097415, WO 2008/010252). Lactic acidbacteria according to Patent Application WO 2008/010252 were used underthe same conditions of lactic acid bacteria according to the presentinvention and were not suitable to gluten degradation, in fact residualgluten content are on the order of ca. 6000-10000 ppm (FIG. 5).Therefore said bacteria can be used only for decontamination of glutentraces but they do not display same performances of bacteria accordingto the present invention.

With respect to the paper by Rizzello et al. (Appl. Environ. Microbiol.73:4499-4507, 2007), the possibility to obtain a complete glutendegradation over remarkably shorter times (18 h compared to 48 h)implies, firstly, a considerable technological advantage making thetransformation process comparable to the most frequent and commonindustrial processes for oven products. A too long (48 h) fermentationprocess, in addition to increase the technological costs, could presenthygienic-sanitary risks. Further a more rapid gluten degradation processunavoidably results in the production of a raw material (flour withcompletely hydrolysed gluten) characterized by a different profile offree amino acids and, therefore, suitable to result in different sensorycharacteristics of gluten-free products compared to a longer processcharacterized by unavoidably different enzymatic kinetics.

It is therefore a specific object of the present invention a mixturecomprising or consisting of Lactobacillus sanfranciscensis DSM22063 andLactobacillus plantarum DSM 22064 lactic acid bacteria. The mixture canfurther comprise fungal proteolytic enzymes, as for example Aspergillusoryzae, Aspergillus niger proteases or mixtures thereof.

It is a further object of the present invention the use of as abovedefined mixture for the complete degradation of the gluten in both breadand durum wheat, barley, rye and oat flours.

The present invention further refers to a process for the preparation ofa liquid flour dough with completely degraded gluten suitable to theproduction of leavened gluten-free products comprising or consisting ofthe following steps:

a) culture propagating of Lactobacillus sanfranciscensis DSM22063 andLactobacillus plantarum DSM 22064 lactic acid bacteria;

b) flour mixing at concentrations of 20-50%, preferably 30%, and waterat concentration of 50-80%, preferably 70%, containing the mixture oftwo bacteria of a) step at cell density of ca. 10⁸ cfu/g;

c) adding one or more fungal proteases each at concentration of 200-500ppm, preferably 400 ppm;

d) fermenting for 8-20 h, preferably 12 h, at 30-37° C.

The process can further comprise a step e) of drying of liquid doughobtained in step d). Among the flours suitable to be used in the processthere are both bread and durum wheat, barley, rye, oat flours ormixtures thereof, preferably bread and durum wheat.

Fungal proteolytic enzymes can be selected from the group consisting ofAspergillus oryzae, Aspergillus niger proteases or mixtures thereof.

Therefore, the invention also refers to a liquid or dried flour doughwherein the gluten is completely degraded according to as above definedprocess.

It is a further object of the invention a mixture comprising orconsisting of as above defined dough in combination with one or morenaturally gluten-free flours, as for example, those selected from thegroup consisting of native maize, white maize, rice, quinoa, teff oramaranth, and buckwheat flours. Particularly, above mentioned flours canbe used according to the following percentages: native maize 5-15%,preferably 10%, white maize 5-15%, preferably 10%, rice, quinoa, teff oramaranth 10-30%, preferably 20%, and buckwheat flour 1-10%, preferably5%, wherein said percentages are expressed by weight based on the totalweight of the flour composition. Other ingredients that can be added tothe formulation for gluten-free baked goods based on detoxified wheatflour are, for example, sugar, butter, egg and animal liquid cream.

It is a further object of the invention a process for the preparation ofleavened baked goods by using of gluten detoxified flour according to asabove defined process comprising or consisting of the following steps:

a) add a mixture of naturally gluten-free flours at 10-40%, preferably30%, baker's yeast 1-2%, salt 0.1-1.0% and structuring agents 0.5-1% togluten detoxified liquid flour dough using as above defined process andknead;

b) allow the fermentation to occur for approximately 1-3 h, preferably1.5 h, at 30° C.;

c) bake for 50 minutes at 220° C. Where gluten detoxified flour dough isdried, the ingredient to water % ratio will be approximately 1.2:0.8.

The naturally gluten-free flours can be selected from the groupconsisting of native maize, white maize, rice, quinoa, teff, amaranth,buckwheat flour or mixtures thereof. On the other hand the glutendetoxified flour can be selected from the group consisting of both breadand durum wheat, barley, rye, oat flour and mixtures thereof, preferablybread or durum wheat flour.

Therefore, it is an object of the present invention also leavened bakedgoods obtainable by means of as above defined process.

A further object of the present invention is the process for thepreparation of leavened baked goods comprising or consisting of thefollowing steps:

a) add directly native maize, rice flour, egg, sugar, butter and baker'syeast to gluten detoxified flour dough according to as above definedprocess and knead;

b) allow the fermentation to occur for 1.5 h at 30° C. and

c) bake the leavened dough for 50 minutes at 250° C.

Particularly, in step a) the ingredient % are as below: native maize10%, rice flour 10%, egg 5%, sugar 3%, butter 1% and baker's yeast 1.5%.

Therefore, the leavened baked goods obtainable by means of as abovedescribed process constitute an object of the present invention.

A further object of the present invention is also the use of theproducts according to the present invention, i.e. flour dough, mixtureof the dough with gluten-free flours, leavened baked goods, leavenedbaked goods suitable to cover nutritional imbalances resulting fromgluten-free diet.

Finally, Lactobacillus sanfranciscensis DSM22063 and Lactobacillusplantarum DSM 22064 lactic acid bacteria represent an object of thepresent invention.

The present invention now will be described by an illustrative, but notlimitative way according to preferred embodiments thereof, withparticular reference to enclosed drawings.

FIG. 1 shows N type aminopeptidase (PepN), dipeptidase (PepV) andtripeptidase (PepT) (a), and proline iminopeptidase (PepI), prolidase(PepQ), prolinase (PepR), dipeptidil-peptidase (PepX) (b) activities ofLactobacillus sanfranciscensis DPPMA12 (DSM22063) and Lactobacillusplantarum DPPMA125 (DSM22064), on Leu-p-NA, Leu-Leu, Leu-Leu-Leu andPro-p-NA, Val-For-Gly and Gly-For-Wing synthetic substrates,respectively. Lactic acid bacteria used in the study of Rizzello et al.(Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507) wereemployed as control: Lactobacillus alimentarius 15M, Lactobacillusbrevis 14G, L. sanfranciscensis 7A, Lactobacillus hilgardii 51B and L.sanfranciscensis LS3, LS10, LS19, LS23, LS38 and LS47. The enzymaticactivity was expressed as activity unit (U), i.e. enzyme amountnecessary to release 1 μmol/min of p-nitroanilide or 1 μmol/min of aminoacid for the activities on substrates different from p-nitroanilide.

FIG. 2 shows bi-dimensional electrophoretic profiles of various durumwheat varieties (Svevo and Duilio) before and after treatment withselected lactic acid bacteria and fungal proteases.

FIG. 3 shows the protein composition of wheat flour before and after thehydrolysis process by selected lactic acid bacteria and fungalproteases.

FIG. 4 shows aminopeptidase (A), proline iminopeptidase (B) andprolyl-dipeptidil aminopeptidase (C) activities of lactic acid bacteriaused according to WO2008/010252 (Lactobacillus sanfranciscensis LS40,LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41) and the presentinvention [L. sanfranciscensis DPPMA12 (DSM22063) and Lactobacillusplantarum DPPMA125 (DSM22064)]. The acronyms 15M, 14G, 7A, 51B, LS3,LS10, LS19 LS23, LS38 and LS47 stand for biotypes as used according toRizzello et al., 2007, publication.

FIG. 5 shows the residual gluten concentration (ppm) in fermented doughsfrom Lactobacillus sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11,LS18, LS4, LS15 and LS41 (WO2008/010252) and from L. sanfranciscensisDPPMA12 (DSM22063) and Lactobacillus plantarum DPPMA125 (DSM22064) for12 h at 37° C.

FIG. 6 shows the concentration of total free amino acids (mg/kg) infermented dough using different combinations of lactic bacteriaaccording to WO2008/010252 (dough 1, 2, 3, 4 and 5) and wheat flourdough fermented with two lactic acid bacteria (DDPPMA12 and DPPMA125) ofthe present invention.

FIG. 7 shows the Principal Component Analysis (PCA) of the data obtainedfrom sensory analysis of breads (1, 2, 4 and 5) according toWO2008/010252 and bread (DPPMA12 and DPPMA125) obtained using detoxifiedwheat flour according to the invention.

EXAMPLE 1 Peptidase Activity of Selected Lactic Bacteria

L. sanfranciscensis DPPMA12 and L. plantarum DPPMA125 from the CultureCollection of the Dipartimento di Protezione delle Piante andMicrobiologia Applicata dell'Universita degli Studi di Bari, previouslyisolated from “sourdoughs”, were propagated at 30° C. for 24 h inmodified MRS (mMRS), containing, in addition to usual ingredients, 5%maltose and 10% yeast water—final pH 5.6. As control for peptidaseactivities of lactic bacteria used in the recent publication by Rizzelloet al. (Rizzello et al., 2007. Appl. Environ. Microbiol. 73:4499-4507)were used: Lactobacillus alimentarius 15M, Lactobacillus brevis 14G, L.sanfranciscensis 7A, Lactobacillus hilgardii 51B and L. sanfranciscensisLS3, LS10, LS19, LS23, LS38 and LS47.

Cells cultivated for 24 h, harvested by centrifugation (10000 rpm, 4°C.), washed twice in phosphate buffer 50 mM, pH 7.0 and re-suspended insame buffer at 2.5 (A₆₂₀ nm) optical density, corresponding to 10⁸cfu/ml, were used for enzyme assays. Type N (PepN) aminopeptidase andproline iminopeptidase (PepI) activities, were determined using Leu-p-NAand Pro-p-NA synthetic substrates, respectively. The reaction mixtureconsisted of: 0.9 ml of K-phosphate buffer 50 mM, pH 7.0 containingdissolved synthetic substrate (final concentration 2 mM) and 100 μl ofcellular suspension. The enzymatic activity, expressed as activity unit(U), corresponds to enzyme amount necessary to release 1 μmol/min ofp-nitroanilide (Gobbetti et al., 1996. The proteolytic system ofLactobacillus sanfranciscensis CB1: purification and characterization ofa proteinase, a dipeptidase, and an aminopeptidase. Appl. Environ.Microbiol. 62: 3220-3226). Prolidase (PepQ), prolinase (PepR) anddipeptidil-peptidase (PepX) were determined as described by Cagno andco-workers, (Di Cagno et al., 2004. Sour dough bread made from wheat andnontoxic flours and starter with selected lactobacilli is tolerated inceliac sprue patients, Appl. Environ. Microbiol. 70: 1088-1096) on,respectively, Val-Pro, Pro-Gly and Gly-Pro-Ala. Dipeptidase (PepV) andtripeptidase (PepT) were determined according to Cd-ninidrine method(Gobbetti et al., 1999. Study of the effects of temperature, pH, NaCl,and aw on the proteolytic and lipolytic activities of cheese-relatedlactic bacteria by quadratic response surface methodology, EnzymeMicrobial Technol 25: 795-809) using, respectively, Leu-Leu andLeu-Leu-Leu. One activity unit (U) is defined as the enzyme amountnecessary to release 1 μmol/min of amino acid.

For comparative purposes, the test was repeated also for the lacticbacteria described in WO2008/010252 (L. sanfranciscensis LS40, LS13,LS44, LS35, LS14, LS11, LS18, LS4, LS15 and LS41).

EXAMPLE 2 Protein Extraction from Wheat Flour and ElectrophoreticAnalysis

Proteins were extracted from wheat flour according to method describedby Weiss et al. (Weiss et al., 1993. Electrophoretic characterization ofwheat grain allergens from different cultivars involved in bakersasthma. Electrophoresis. 14:805-816). Bi-dimensional electrophoreticanalysis of ca. 30 μg of extracted fraction protein was carried outaccording to immobiline-polyacrilamide method (De Angelis et al., 2005.Biochim. Biophys. Acta. 1762:80-93). Four gels for each independentfermentation were analyzed and data were normalized according toprocedure as proposed by Bini et al. (Bini et al., 1997. Proteinexpression profiles in human breast ductal carcinoma and histologicallynormal tissue. Electrophoresis. 18:2831-2841).

EXAMPLE 3 Immunological and Mass Spectrometry MALDI-TOF Analyses

Immunological analyses were carried out by using of R5 antibody, andsandwich and competitive ELISA test (Transia Plate, Diffchamb) (Valdezet al., 2003. Innovative approach to low-level gluten determination infoods using sandwich enzyme-linked immunosorbent assay protocol. Eur. J.Gastroenterol. Hepatol. 15:465-474). MALDI-TOF spectrometry analysis wascarried using Voyager-De Pro-workstation (PerSeptive Biosystems UnitedKingdom) according to method reported by Hernando et al. (Hernando etal., 2003. New strategy for the determination of gliadin in maize orrice-based foods matrix-assisted laser desorption/ionizationtime-of-flight mass spectrometry fractionation of gliadin from maize orrice-prolamins by acid treatment. J. Mass Spectrom. 38:862-871).

Protein concentration was determined according to Bradford method(Bradford, 1976. A rapid and sensitive method for the quantification ofmicrogram quanties of protein utilizing the principle of protein-dyebinding. Anal. Biochem. 72:248-254). Organic nitrogen concentration wasdetermined according to Kjeldahl method. Free amino acid concentrationwas determined using an amino acid analyzer (Biochrom Ltd., CambridgeScience Park, United Kingdom) (Di Cagno et al., 2004. Appl. Environ.Microbiol. 70:1088-1096).

For comparative purposes, free amino acid concentration was determinedalso in dough obtained using lattobacilli described in WO2008/010252 (L.sanfranciscensis LS40, LS13, LS44, LS35, LS14, LS11, LS18, LS4, LS15 andLS41) after fermentation for 24 hours at 30° C. according to proceduresas indicated in the protocol reported in FIG. 8 of said document.

EXAMPLE 4 Manufacture of Leavened Baked Goods Using Detoxified WheatFlour

Cultures of two selected lactic acid bacteria were propagated in culturemedium, washed and re-suspended in water as previously described. Wheatflour was mixed at 30% with water (70%) containing the mixture of saidtwo lactic acid bacteria at a cell density of ca. 10⁸ cfu/g, and fungalenzymes, each at 400 ppm concentration, were added. Fermentation wascarried out for 12 h at 37° C. After fermentation, directly to liquiddough native maize (10%), rice flour (10%), egg (5%), sugar (3%), butter(1%) and baker's yeast (1.5%) were added. The concentrations are basedon the total dough weight. After kneading, the fermentation is allowedfor 1.5 h at 30° C., before the baking of leavened dough for 50 minutesat 250° C.

The process can further comprise a drying step of liquid wheat flourdough. Different ingredients were also used for the production ofgluten-free bread.

For comparison purposes also breads 1, 2, 4 and 5 were producedaccording to the protocol of Patent Application WO2008/010252. Sensoryanalysis of breads obtained according to the invention and known art,respectively, was carried out, particularly the following descriptorswere considered: elasticity, acid fragrance, acid taste, sweetness,dryness and fragrance. Each descriptor was evaluated according to ascore scale from 0 to 100. The results of sensory analysis wereprocessed by Principal Component Analysis. Moreover, the breads wereanalyzed for specific volume, crumb structure, stiffness and fibrecontent according to standard methods of the American Association ofCereal Chemistry (AACC).

EXAMPLE 5 Administration to Celiac Patients of Leavened Baked Goods Madeof Detoxified Wheat Flour

Baked goods based on detoxified wheat flour, obtained as previouslydescribed, were administered to 5 celiac patients. Opportunely theceliac pathology diagnosis has been acquired according to criteria asproposed by the European Society for Pediatric Gastroenterology,Hepatology and Nutrition. Patient average age was ca. 15 years. Celiacpatients were under remission conditions since at least two years andsubjected to a controlled gluten-free diet. All patients at recruitmentshowed serological pathology negative indicators, as well as negativehistochemical assays. Each patient, during a 60 day period, dailyconsumed baked goods which contained detoxified wheat flourcorresponding to 10 g of native gluten equivalent. Immunochemical andhistological assays were carried out at the Dipartimento di Pediatria eGastroeneterologia dell'Universita degli Studi di Napoli, Federico II.Patient recruitment occurred with informed consent of the parents towhich the experimental schedule, previously approved by the EthicalCommittee of the University of Naples, has been subjected.

Results

(1) Peptidase Activity of Selected Lactic Acid Bacteria

Peptidase activity was assayed on synthetic substrates relativelyspecific for peptidase activities which are important for thedegradation of gluten derived oligopeptides (FIG. 1). It is possible toobserve that L. sanfranciscensis DPPMA12 and L. plantarum DPPMA125display all the considered enzymatic activities. With the exception oftripeptidase (PepT) type activity, two selected lactic acid bacteria,and particularly L. plantarum DPPMA125, display values for otherpeptidase activities significantly (P<0.05) higher than biotypes used inthe study by Rizzello et al. (Rizzello et al., 2007. Appl. Environ.Microbiol. 73:4499-4507). Significant differences, particularly forPepI, PepQ, PepR and PepX activities, with the presence of prolineresidues at various bond positions, were detected. Glutenin and inparticular gliadins, contain a very high and unusual percentage (45-60%)of glutamine and proline residues. This last iminoacid, consequently,particularly occurs in toxic epitopes, resulting from wheat flour, andresponsible of celiac pathology. To provide for microorganisms suitablefor high degradation of bond where proline is involved in, this enzymeactivity is certainly a pre-requirement for intense gluten degradationand rapid hydrolysis process. The availability of a large CultureCollection to be screened and the large number of assayed enzymaticactivities represent, therefore, the requirement to get not commonlyavailable selected strains. Peptidase activity of selected lacticbacteria is enhanced by the complementary use of fungal proteasesroutinely used in bread-making processes. Such enzymes are employed inthe bread-making industry in order to modify the protein concentrationand, therefore, the “flour strength”, depending on baked goods they aredesigned to.

FIG. 4 shows a comparison of peptidase activities for known art lacticacid bacteria and two lactic acid bacteria (DPPMA12 and DPPMA125)according to the invention, respectively, and it is apparent that thelatter display markedly higher aminopeptidase, proline iminopeptidaseand prolyl-dipeptidil aminopeptidase activities.

(2) Characterization of Hydrolised Flour

After fermentation for 12 h at 37° C., protein fractions wereselectively extracted and subjected to complementary analytical assays.As it is apparent using bi-dimensional electrophoretic analysis (FIG.2), at the end of fermentation process no traces of gliadins from Svevoand Duilio durum wheat variety flours are detectable. Similar resultswere found in the glutenin fraction for bread commercially available“00” type wheat, other durum wheat tested varieties (Arcangelo, Ciccio,Colosseo, Gargano and Simeto) and barley, rye and oat flours. Wheatflour proteins, extracted with 60% ethanol, were analyzed with MALDI-TOFMS technique. Peaks corresponding to gliadin European standardcompletely disappeared after fermentation for 12 h at 37° C. Only somepeaks with molecular mass lower than 8 kDa were detected by spectrometryanalysis. Immunological analyses carried out using R5 antibodies andELISA assays confirmed that no gliadin traces were detectable infermented sample. According to same method the residual glutenconcentration determined in bread commercially available type “00”wheat, durum wheat variety and barley, rye and oat flours was, in allthe cases, lower than 20 ppm. The method used for these determinationsis an AIC (Associazione Italiana Celiachia), WHO and FAO officialmethod. With respect to literature reported data (Rizzello et al., 2007.Appl. Environ. Microbiol. 73:4499-4507), the hydrolysis process iscarried out with the same effectiveness (residual gluten <20 ppm) but ina markedly shorter times (12 versus 48 h). This process high rateresults, on the one hand, from the use of higher concentration of eachof fungal proteolytic enzymes (400 ppm) and, on the other hand, mainlyfrom higher peptidase activity of selected lactic bacteria biotypes.Again in comparison to said literature reference which only consideredbread wheat flour with low initial gluten concentration, the presentinvention shows the protocol effectiveness also on various durum wheatvarieties, and barley, rye and oat flours having also initial elevatedprotein concentrations.

In FIG. 3 organic nitrogen content of bread commercially available type“OO” wheat flour before and after the fermentation process is reported.Hydrolysed flour almost totally consists of a mixture of low molecularweight peptides and amino acids. Only an amount lower than 20% ofinitial glutenins is yet present in hydrolysed flour. Amino acidconcentration in hydrolysed flour is about 15000 mg/kg compared to<1.000 mg/kg occurring in wheat flour. Higher bioavailability of freeamino acids makes this hydrolysed wheat flour a raw material with highnutritional content, retaining at the same time other cereal nutritionalcharacteristics in terms of mineral salts, vitamins and fibres. Whereused as an ingredient for the production of gluten-free foods, thehydrolysed wheat flour would cover nutritional imbalances resulting fromgluten-free diet (Grehn et al., 2001. Scand J Nutr 45: 178-182; Marianiet al., 1998. J Pediart Gastroenterol Nut 27: 519-523; Thompson et al.,2005. J. Human. Nutr. Diet. 18:163-169).

Comparative test with known art lactic acid bacteria shows that theamino acid concentration in the dough is remarkably lower and equal toabout 2000 mg/kg after highest hydrolysis condition (FIG. 6). Thisconfirms the different hydrolysis degree of wheat proteins. Moreover,since released amino acids are precursors of volatile compounds whichare generated during baking process and are responsible for baked goodstaste, remarkably higher free amino acid concentration, as in the caseof the present invention, indicates higher synthesis of volatilecompounds and, therefore, a better taste of the products according tothe invention.

(3) Production of Leavened Baked Goods Using Detoxified Wheat Flour

An application example of biotechnological protocol for the manufactureof leavened baked goods based on detoxified wheat flour was reportedabove. In addition to the manufacture of baked goods, the protocol wasstandardized and optimized also for gluten-free bread production withpreviously described ingredients. In addition to the possible direct useof detoxified wheat flour, a treatment thereof using spry-drier andsubsequent use as dried matter is possible. This further technologicalpossibility allows an easy conservation of the raw material over thetime, without any alteration of wheat flour nutritional characteristics.

FIG. 7 shows the best sensory properties of the bread according to thepresent invention (DPPMA12+DPPMA125) compared to known art breads.Moreover, table 1 shows that the bread according to the presentinvention is characterized by higher specific volume, more crumbbubbles, lower stiffness, and higher fibre content compared to known artbread. These differences result from the presence of wheat flour thatalthough detoxified is suitable to favour better rheological andchemical characteristics.

TABLE 1 Rheological and Bread Bread chemical parameters Starter 1*(DPPMA12 + DPPMA125) Specific volume 1.35 ± 0.04 1.48 ± 0.07 (cm3/g)Crumb bubbles (%) 39.2 ± 0.34 42.3 ± 0.47 Stiffness (n) 16.62 ± 0.27 14.75 ± 0.21  Fibre content (%)  1.5 ± 0.44  2.0 ± 0.52 *Lactobacillussanfranciscensis DSM18426, DSM18427, Lactobacillus plantarum DSM18430

(4) Administration to Celiac Patients of Baked Goods Made of DetoxifiedWheat Flour

Baked goods prepared according to previously described biotechnologicalprotocol have been administered daily to celiac patients correspondingto a dose equivalent to 10 g of native gluten. In Table 2 immunochemicaland histological indices of in remission celiac patients subjected toconsumption (10 g of gluten equivalent to the day for 60 gg) based ondetoxified wheat flour are reported.

TABLE 2 Immunochemistry Anti-tTG CD3 CD25 γδ Marsh Grade T₀ T₆₀ T₀ T₆₀T₀ T₆₀ T₀ T₆₀ T₀ T₆₀ F.I. 1.6 1 39 38 6 5 5.6 8.6 0 0 I.C. 1.9 1.1 3.711 11 9 0.9 3.8 0 0 R.R. 0.3 0.3 53 56 3 4 11.5 17.8 1 1 I.I. 0.5 0.3 3136 21 21 8.4 12.8 0 0

As it is possible to observe, all the patients at recruitment time (T₀)display normal serological and histological values (Marshes Grade).After each daily assumption of 10 g gluten equivalent, over a 60 dayperiod (T₆₀) none of biochemical and immunohistochemical values wasdifferent compared to initial value. In particular, it is to be observedthat Marsh Grade, who represents the integrity condition andfunctionality of the intestinal mucosa, as detected based on bio-opticalsample, is absolutely identical to initial value. No patient developedatrophy of intestinal villas during the challenge. Only one out 5recruited patients interrupted the test due to personal reasons and notdepending on eventual pathological condition. On the base of acquiredresults based on most careful in vivo clinical analyses, it is possibleto state that the detoxified wheat flour was tolerated by all thepatients. In conclusion, detoxified wheat flour can be used for thepreparation of gluten-free foods.

The invention claimed is:
 1. Mixture comprising or consisting ofLactobacillus sanfranciscensis DSM22063 and Lactobacillus plantarum DSM22064 lactic acid bacteria, wherein the lactic acid bacteria each havean x-prolyl dipeptidyl aminopeptidase activity of between 17.5 U and 22U, U being defined as the enzyme amount necessary to release 1 μmol/minof amino acid.
 2. Mixture according to claim 1, further comprisingfungal proteases.
 3. Mixture according to claim 2, wherein fungalproteases are selected from the group consisting of Aspergillus oryzae,Aspergillus niger proteases or mixtures thereof.
 4. A mixturecomprising: a) Lactobacillus sanfranciscensis DSM22063 and Lactobacillusplantarum DSM 22064 lactic acid bacteria; b) water; and c) flour;wherein the lactic acid bacteria each have an x-prolyl dipeptidylaminopeptidase activity of between 17.5 U and 22 U, U being defined asthe enzyme amount necessary to release 1 μmol/min of amino acid; andwherein the flour is at least one gluten-containing flour selected fromthe group consisting of bread wheat, durum wheat, tender wheat, barley,rye and oat flours.